Letters in Applied Microbiology ISSN 0266-8254
Editor’s Choice
ORIGINAL ARTICLE
Suppression of Tomato mosaic virus disease in tomato plants by deep ultraviolet irradiation using light-emitting diodes S. Matsuura and S. Ishikura Agriculture Research Center, Hiroshima Prefectural Technology Research Institute, Hiroshima, Japan
Significance and Impact of the Study: Disease caused in tomato plants by resistance-breaking Tomato mosaic virus (ToMV) could be suppressed by ultraviolet (UV)-B irradiation using light-emitting diodes (LEDs). This paves the way for the future management of plant viral diseases using deep UV LEDs.
Keywords deep UV, light-emitting diodes, suppression, tomato, Tomato mosaic virus, UV-B. Correspondence Shohei Matsuura, Agriculture Research Center, Hiroshima Prefectural Technology Research Institute, Hara 6869, Higashihiroshima, Hiroshima 739-0151, Japan. E-mail: [email protected] 2014/0789: received 16 April 2014, revised 12 June 2014 and accepted 24 June 2014
Abstract Resistance-breaking strains of Tomato mosaic virus (ToMV) are emerging in many countries, including Japan. We examined whether deep ultraviolet (UV) irradiation on tomato plants using light-emitting diodes (LEDs) could suppress the expression of ToMV symptoms. We also investigated the optimum wavelength and radiant exposure for suppressing the disease effectively in tomato plants. Among the three wavelengths tested, UV irradiation at 280– 290 nm had a relatively high suppressive effect on ToMV and resulted in a low incidence of UV damage. Pre-inoculation exposure to UV was effective in suppressing viral disease, indicating that acquired resistance was induced by UV irradiation. UV-B fluence of 07–14 kJ m2 day1 at wavelengths of 280– 290 nm suppressed ToMV effectively without significant UV damage.
doi:10.1111/lam.12301
Introduction Tomato mosaic virus (ToMV) is a positive-sense ssRNA virus which belongs to the genus Tobamovirus. ToMV causes serious loss of yield and fruit quality in tomato (Solanum lycopersicum). Three resistance genes (Tm-1, Tm-2 and Tm-2a) from wild Solanum species have been introduced into commercial tomato cultivars to control infection by ToMV. In the tissues of tomato plants carrying these genes, multiplication or movement of ToMV and Tobacco mosaic virus (TMV) is inhibited. However, resistance-breaking ToMV strains have emerged to cause severe symptoms such as mosaic and necrosis in resistant tomato cultivars (Harrison 2002). Several amino acid exchanges in the 130/180-kDa proteins (for Tm-1) or the 30-kDa movement protein (for Tm-2 and Tm-2a) are involved in the breaking of resistance (Meshi et al. 1988, 1989; Weber et al. 1993; Strasser and Pfitzner 2007). In Japan, the emergence of resistance-breaking ToMV strains has been increasing in the past few years, threatening the stability of tomato production. Novel innovative control
measures therefore need to be developed to control resistance-breaking strains of ToMV. Ultraviolet (UV) is divided into UV-A (315–400 nm), UV-B (280–315 nm) and UV-C (100–280 nm), and wavelengths below 300 nm are defined as deep UV. To date, suppression of several fungal diseases by UV-B irradiation has been reported (Kanto et al. 2009; Suthaparan et al. 2012). Exposure of tobacco (Nicotiana tabacum) to UV-C induces the accumulation of salicylic acid and pathogenesis-related proteins and increases resistance to TMV (Brederode et al. 1991; Yalpani et al. 1994). In addition, Kobayashi et al. (2012) recently reported that irradiation with UV-B suppresses the accumulation of Tomato spotted wilt virus (TSWV) in tobacco plants, as well as the incidence of the disease. It is therefore possible that UV irradiation can induce defence responses to tomato plants and suppress the disease caused by resistance-breaking ToMV strains. Since the 1990s, substantial progress has been made in shortening the wavelength and increasing the UV power of light-emitting diodes (LEDs) using nitride materials
Letters in Applied Microbiology 59, 457--463 © 2014 The Society for Applied Microbiology
457
Suppression of ToMV using deep UV LEDs
S. Matsuura and S. Ishikura
such as AlGaN compounds (Hirayama 2005), thus enabling these LEDs to be used in many applications such as sterilization, water purification and medical treatment. LEDs have an advantage over low-pressure mercury lamps in that emissions of particular narrow, half-width wavelengths can be selected freely. Therefore, specific, desirable wavelengths can be used with high energy efficiency. Our aim here was to determine whether deep UV irradiation using LEDs could suppress the incidence of ToMV. We also aimed to identify the optimum UV wavelength and radiant exposure, with the ultimate goal of developing deep UV LED irradiation as a control measure for resistance-breaking strains of ToMV in tomato. Results and discussion Suppressive effects of each UV wavelength We used LEDs of different wavelengths to evaluate the suppression of ToMV disease by UV irradiation. In nonUV-irradiated plants, severe yellowing and necrotic spots occurred on the upper leaves (third and fourth true leaves) of the tomato plants, which showed dwarfing 7 days after virus inoculation (Fig. 1c). In contrast, disease severity was significantly lower in plants irradiated
(a)
(b)
(c)
(d)
with UV at wavelengths of 290 nm or less than in the non-UV-irradiated controls (P < 001) (Figs 1a,b and 2a). Disease severity in plants irradiated with UV at 295– 305 nm was similar to that in non-UV-irradiated controls (Fig. 2a). The amount of target ToMV RNA per total RNA (lg) was significantly lower in plants irradiated with UV at wavelengths of 290 nm or less than in non-UVirradiated controls (P < 0001) (Fig. 2b). In contrast, the amount of viral RNA in plants irradiated with UV at 295–305 nm was not significantly different from that in the non-UV-irradiated controls (Fig. 2b). Severe UV damage, with leaf curl and size reduction, was observed on plants irradiated with UV at 260–270 nm; the UV damage caused by UV irradiation at 280–290 nm was milder than that caused by irradiation at 260–270 nm (Fig. 1a,b). Here, among the three wavelengths used, irradiation with UV at 290 nm or less significantly suppressed symptom expression caused on tomato plants by a resistancebreaking ToMV strain. In contrast, UV at wavelengths of 295 nm or more hardly suppressed viral disease on the tomato plants. UV at wavelengths of 290 nm or less significantly reduced virus concentrations compared with those in non-UV-irradiated controls, indicating that suppression of symptom expression was due to a reduction
Figure 1 Disease severity and UV damage on plants irradiated with deep UV emitted from LEDs. (a) UV at wavelengths of 260–270 nm and radiant exposure of 720 J m2 day1 with ToMV inoculation, (b) UV at 280– 290 nm and 1440 J m2 day1 with ToMV inoculation, (c) non-UV-irradiated control with ToMV inoculation, (d) mock (non-UVirradiated control without ToMV inoculation).
458
Letters in Applied Microbiology 59, 457--463 © 2014 The Society for Applied Microbiology
S. Matsuura and S. Ishikura
Suppression of ToMV using deep UV LEDs
Disease severity (0–4)
a
a
3
2
1 b 0
b
260–270
280–290 295–305 Wavelength (nm)
Viral RNA (pg µg–1 of total RNA)
(b) 3·0
(a) 4
a
2·0 1·5 1·0 0·5
b
b 0·0
Control
a
2·5
260–270
280–290 295–305 Wavelength (nm)
Control
Figure 2 Effects of various wavelengths of deep UV emitted from LEDs on disease severity (a) and accumulation of ToMV (b) in tomato plants. UV irradiation of tomato plants was started 3 days before virus inoculation, and then, the plants were irradiated with UV for 7 days. UV radiant exposure was set to 1440 J m2 day1 (720 J m2 day1 for LEDs emitting wavelengths 260–270 nm) at the plant surface. Control indicates no UV irradiation. Vertical bars indicate standard errors. Means followed by different letters under disease severity and virus amount are significantly different by Kruskal–Wallis test and Tukey’s test, respectively (n = 5, P < 005).
in virus multiplication in the UV-irradiated tomato plants. UV damage (weight reduction and leaf curl) was observed after UV irradiation at wavelengths of 290 nm or less and was particularly severe in the wavelength range of 260–270 nm. Therefore, LED modules at wavelengths of 280–290 nm rather than 260–270 nm are suitable for use in suppressing ToMV disease in tomato plants. Pre- and post-inoculation UV irradiation We evaluated the effects of UV irradiation timing on the incidence of ToMV disease in tomato plants. Disease severity was significantly lower in plants irradiated with
Disease severity (0–4)
3 a 2 b 1 b
Pre inoculation
Post inoculation UV-B irradiation timing
Viral RNA (pg µg–1 of total RNA)
(b) 2·0
(a) 4
0
UV (280–290 nm) than in non-UV-irradiated controls, regardless of the timing of irradiation (P < 001) (Fig. 3a). The amounts of viral RNA in plants irradiated with UV (280–290 nm) before (P < 0001) and after (P < 001) virus inoculation were significantly lower than in non-UV-irradiated controls (Fig. 3b). We found here that the suppressive effect of ToMV was high even when tomato plants were irradiated with UV-B only before inoculation. These results suggested that acquired resistance to ToMV in tomato plants was induced in advance by UV irradiation. The mechanism of virus suppression in tomato plants by UV irradiation may mainly be a reduction in ToMV multiplication, because
Control
1·5 a 1·0
b
0·5 b 0·0
Pre inoculation
Post inoculation UV-B irradiation timing
Control
Figure 3 Effects of UV-B irradiation of tomato plants before or after virus inoculation on disease severity (a) and accumulation of ToMV (b). Tomato plants were irradiated with UV at wavelengths of 280–290 nm and a radiant exposure of 1440 J m2 day1. Pre-inoculation indicates that plants were irradiated with UV for 3 days before inoculation. Post-inoculation indicates that plants were irradiated with UV for 7 days after inoculation. Control indicates no UV irradiation. Vertical bars indicate standard errors. Means followed by different letters under disease severity and virus amount are significantly different by Kruskal–Wallis test and Tukey’s test, respectively (n = 7, P < 005).
Letters in Applied Microbiology 59, 457--463 © 2014 The Society for Applied Microbiology
459
Suppression of ToMV using deep UV LEDs
S. Matsuura and S. Ishikura
suppression of ToMV was also observed upon UV irradiation after virus inoculation. The suppressive effect observed when the plants were UV irradiated after inoculation was slightly inferior to the effect when irradiation occurred pre-inoculation. Therefore, exposure to UV-B before virus infection is an important strategy for controlling viral diseases. RNA silencing operates as an innate plant defence system against viruses in higher plants (Voinnet 2001). It would be interesting to determine whether the induction of acquired resistance in tomato plants by UV irradiation involves RNA silencing. Further research is needed to elucidate the mode of action of ToMV suppression by deep UV irradiation in tomato plants. Optimum UV radiant exposures We evaluated the optimum UV radiant exposures for suppressing ToMV disease in tomato plants. Disease severity and the amount of viral RNA were both negatively correlated (P < 00001) with UV radiant exposure (Fig. 4). The amount of viral RNA was reduced markedly (to less than about 1 pg lg1 of total RNA) at radiant exposures of 720 J m2 day1 or more (Fig. 4b). The UV damage in tomato plants irradiated with UV at wavelengths of 280–290 nm with various radiant exposures for 7 days is shown in Table 1. There were no significant differences in weight of the above-ground parts of plants among the radiant exposures (P = 03359). Although distinct or severe leaf curl was observed in tomato plants irradiated with UV at 1440 J m2 day1, little or no leaf curl was observed at
Editor’s Choice
ORIGINAL ARTICLE
Suppression of Tomato mosaic virus disease in tomato plants by deep ultraviolet irradiation using light-emitting diodes S. Matsuura and S. Ishikura Agriculture Research Center, Hiroshima Prefectural Technology Research Institute, Hiroshima, Japan
Significance and Impact of the Study: Disease caused in tomato plants by resistance-breaking Tomato mosaic virus (ToMV) could be suppressed by ultraviolet (UV)-B irradiation using light-emitting diodes (LEDs). This paves the way for the future management of plant viral diseases using deep UV LEDs.
Keywords deep UV, light-emitting diodes, suppression, tomato, Tomato mosaic virus, UV-B. Correspondence Shohei Matsuura, Agriculture Research Center, Hiroshima Prefectural Technology Research Institute, Hara 6869, Higashihiroshima, Hiroshima 739-0151, Japan. E-mail: [email protected] 2014/0789: received 16 April 2014, revised 12 June 2014 and accepted 24 June 2014
Abstract Resistance-breaking strains of Tomato mosaic virus (ToMV) are emerging in many countries, including Japan. We examined whether deep ultraviolet (UV) irradiation on tomato plants using light-emitting diodes (LEDs) could suppress the expression of ToMV symptoms. We also investigated the optimum wavelength and radiant exposure for suppressing the disease effectively in tomato plants. Among the three wavelengths tested, UV irradiation at 280– 290 nm had a relatively high suppressive effect on ToMV and resulted in a low incidence of UV damage. Pre-inoculation exposure to UV was effective in suppressing viral disease, indicating that acquired resistance was induced by UV irradiation. UV-B fluence of 07–14 kJ m2 day1 at wavelengths of 280– 290 nm suppressed ToMV effectively without significant UV damage.
doi:10.1111/lam.12301
Introduction Tomato mosaic virus (ToMV) is a positive-sense ssRNA virus which belongs to the genus Tobamovirus. ToMV causes serious loss of yield and fruit quality in tomato (Solanum lycopersicum). Three resistance genes (Tm-1, Tm-2 and Tm-2a) from wild Solanum species have been introduced into commercial tomato cultivars to control infection by ToMV. In the tissues of tomato plants carrying these genes, multiplication or movement of ToMV and Tobacco mosaic virus (TMV) is inhibited. However, resistance-breaking ToMV strains have emerged to cause severe symptoms such as mosaic and necrosis in resistant tomato cultivars (Harrison 2002). Several amino acid exchanges in the 130/180-kDa proteins (for Tm-1) or the 30-kDa movement protein (for Tm-2 and Tm-2a) are involved in the breaking of resistance (Meshi et al. 1988, 1989; Weber et al. 1993; Strasser and Pfitzner 2007). In Japan, the emergence of resistance-breaking ToMV strains has been increasing in the past few years, threatening the stability of tomato production. Novel innovative control
measures therefore need to be developed to control resistance-breaking strains of ToMV. Ultraviolet (UV) is divided into UV-A (315–400 nm), UV-B (280–315 nm) and UV-C (100–280 nm), and wavelengths below 300 nm are defined as deep UV. To date, suppression of several fungal diseases by UV-B irradiation has been reported (Kanto et al. 2009; Suthaparan et al. 2012). Exposure of tobacco (Nicotiana tabacum) to UV-C induces the accumulation of salicylic acid and pathogenesis-related proteins and increases resistance to TMV (Brederode et al. 1991; Yalpani et al. 1994). In addition, Kobayashi et al. (2012) recently reported that irradiation with UV-B suppresses the accumulation of Tomato spotted wilt virus (TSWV) in tobacco plants, as well as the incidence of the disease. It is therefore possible that UV irradiation can induce defence responses to tomato plants and suppress the disease caused by resistance-breaking ToMV strains. Since the 1990s, substantial progress has been made in shortening the wavelength and increasing the UV power of light-emitting diodes (LEDs) using nitride materials
Letters in Applied Microbiology 59, 457--463 © 2014 The Society for Applied Microbiology
457
Suppression of ToMV using deep UV LEDs
S. Matsuura and S. Ishikura
such as AlGaN compounds (Hirayama 2005), thus enabling these LEDs to be used in many applications such as sterilization, water purification and medical treatment. LEDs have an advantage over low-pressure mercury lamps in that emissions of particular narrow, half-width wavelengths can be selected freely. Therefore, specific, desirable wavelengths can be used with high energy efficiency. Our aim here was to determine whether deep UV irradiation using LEDs could suppress the incidence of ToMV. We also aimed to identify the optimum UV wavelength and radiant exposure, with the ultimate goal of developing deep UV LED irradiation as a control measure for resistance-breaking strains of ToMV in tomato. Results and discussion Suppressive effects of each UV wavelength We used LEDs of different wavelengths to evaluate the suppression of ToMV disease by UV irradiation. In nonUV-irradiated plants, severe yellowing and necrotic spots occurred on the upper leaves (third and fourth true leaves) of the tomato plants, which showed dwarfing 7 days after virus inoculation (Fig. 1c). In contrast, disease severity was significantly lower in plants irradiated
(a)
(b)
(c)
(d)
with UV at wavelengths of 290 nm or less than in the non-UV-irradiated controls (P < 001) (Figs 1a,b and 2a). Disease severity in plants irradiated with UV at 295– 305 nm was similar to that in non-UV-irradiated controls (Fig. 2a). The amount of target ToMV RNA per total RNA (lg) was significantly lower in plants irradiated with UV at wavelengths of 290 nm or less than in non-UVirradiated controls (P < 0001) (Fig. 2b). In contrast, the amount of viral RNA in plants irradiated with UV at 295–305 nm was not significantly different from that in the non-UV-irradiated controls (Fig. 2b). Severe UV damage, with leaf curl and size reduction, was observed on plants irradiated with UV at 260–270 nm; the UV damage caused by UV irradiation at 280–290 nm was milder than that caused by irradiation at 260–270 nm (Fig. 1a,b). Here, among the three wavelengths used, irradiation with UV at 290 nm or less significantly suppressed symptom expression caused on tomato plants by a resistancebreaking ToMV strain. In contrast, UV at wavelengths of 295 nm or more hardly suppressed viral disease on the tomato plants. UV at wavelengths of 290 nm or less significantly reduced virus concentrations compared with those in non-UV-irradiated controls, indicating that suppression of symptom expression was due to a reduction
Figure 1 Disease severity and UV damage on plants irradiated with deep UV emitted from LEDs. (a) UV at wavelengths of 260–270 nm and radiant exposure of 720 J m2 day1 with ToMV inoculation, (b) UV at 280– 290 nm and 1440 J m2 day1 with ToMV inoculation, (c) non-UV-irradiated control with ToMV inoculation, (d) mock (non-UVirradiated control without ToMV inoculation).
458
Letters in Applied Microbiology 59, 457--463 © 2014 The Society for Applied Microbiology
S. Matsuura and S. Ishikura
Suppression of ToMV using deep UV LEDs
Disease severity (0–4)
a
a
3
2
1 b 0
b
260–270
280–290 295–305 Wavelength (nm)
Viral RNA (pg µg–1 of total RNA)
(b) 3·0
(a) 4
a
2·0 1·5 1·0 0·5
b
b 0·0
Control
a
2·5
260–270
280–290 295–305 Wavelength (nm)
Control
Figure 2 Effects of various wavelengths of deep UV emitted from LEDs on disease severity (a) and accumulation of ToMV (b) in tomato plants. UV irradiation of tomato plants was started 3 days before virus inoculation, and then, the plants were irradiated with UV for 7 days. UV radiant exposure was set to 1440 J m2 day1 (720 J m2 day1 for LEDs emitting wavelengths 260–270 nm) at the plant surface. Control indicates no UV irradiation. Vertical bars indicate standard errors. Means followed by different letters under disease severity and virus amount are significantly different by Kruskal–Wallis test and Tukey’s test, respectively (n = 5, P < 005).
in virus multiplication in the UV-irradiated tomato plants. UV damage (weight reduction and leaf curl) was observed after UV irradiation at wavelengths of 290 nm or less and was particularly severe in the wavelength range of 260–270 nm. Therefore, LED modules at wavelengths of 280–290 nm rather than 260–270 nm are suitable for use in suppressing ToMV disease in tomato plants. Pre- and post-inoculation UV irradiation We evaluated the effects of UV irradiation timing on the incidence of ToMV disease in tomato plants. Disease severity was significantly lower in plants irradiated with
Disease severity (0–4)
3 a 2 b 1 b
Pre inoculation
Post inoculation UV-B irradiation timing
Viral RNA (pg µg–1 of total RNA)
(b) 2·0
(a) 4
0
UV (280–290 nm) than in non-UV-irradiated controls, regardless of the timing of irradiation (P < 001) (Fig. 3a). The amounts of viral RNA in plants irradiated with UV (280–290 nm) before (P < 0001) and after (P < 001) virus inoculation were significantly lower than in non-UV-irradiated controls (Fig. 3b). We found here that the suppressive effect of ToMV was high even when tomato plants were irradiated with UV-B only before inoculation. These results suggested that acquired resistance to ToMV in tomato plants was induced in advance by UV irradiation. The mechanism of virus suppression in tomato plants by UV irradiation may mainly be a reduction in ToMV multiplication, because
Control
1·5 a 1·0
b
0·5 b 0·0
Pre inoculation
Post inoculation UV-B irradiation timing
Control
Figure 3 Effects of UV-B irradiation of tomato plants before or after virus inoculation on disease severity (a) and accumulation of ToMV (b). Tomato plants were irradiated with UV at wavelengths of 280–290 nm and a radiant exposure of 1440 J m2 day1. Pre-inoculation indicates that plants were irradiated with UV for 3 days before inoculation. Post-inoculation indicates that plants were irradiated with UV for 7 days after inoculation. Control indicates no UV irradiation. Vertical bars indicate standard errors. Means followed by different letters under disease severity and virus amount are significantly different by Kruskal–Wallis test and Tukey’s test, respectively (n = 7, P < 005).
Letters in Applied Microbiology 59, 457--463 © 2014 The Society for Applied Microbiology
459
Suppression of ToMV using deep UV LEDs
S. Matsuura and S. Ishikura
suppression of ToMV was also observed upon UV irradiation after virus inoculation. The suppressive effect observed when the plants were UV irradiated after inoculation was slightly inferior to the effect when irradiation occurred pre-inoculation. Therefore, exposure to UV-B before virus infection is an important strategy for controlling viral diseases. RNA silencing operates as an innate plant defence system against viruses in higher plants (Voinnet 2001). It would be interesting to determine whether the induction of acquired resistance in tomato plants by UV irradiation involves RNA silencing. Further research is needed to elucidate the mode of action of ToMV suppression by deep UV irradiation in tomato plants. Optimum UV radiant exposures We evaluated the optimum UV radiant exposures for suppressing ToMV disease in tomato plants. Disease severity and the amount of viral RNA were both negatively correlated (P < 00001) with UV radiant exposure (Fig. 4). The amount of viral RNA was reduced markedly (to less than about 1 pg lg1 of total RNA) at radiant exposures of 720 J m2 day1 or more (Fig. 4b). The UV damage in tomato plants irradiated with UV at wavelengths of 280–290 nm with various radiant exposures for 7 days is shown in Table 1. There were no significant differences in weight of the above-ground parts of plants among the radiant exposures (P = 03359). Although distinct or severe leaf curl was observed in tomato plants irradiated with UV at 1440 J m2 day1, little or no leaf curl was observed at
